Alzheimer's disease is a common, chronic neurodegenerative disease, characterized by a progressive loss of memory and sometimes-severe behavioral abnormalities, as well as an impairment of other cognitive functions that often leads to dementia and death. It ranks as the fourth leading cause of death in industrialized societies after heart disease, cancer, and stroke. The incidence of Alzheimer's disease is high, with an estimated 2.5 to 4 million patients affected in the United States and perhaps 17 to 25 million worldwide. Moreover, the number of sufferers is expected to grow as the population ages.
A characteristic feature of Alzheimer's disease is the presence of large numbers of insoluble deposits, known as amyloid plaques, in the brains of those affected. Autopsies have shown that amyloid plaques are found in the brains of virtually all Alzheimer's patients and that the degree of amyloid plaque deposition correlates with the degree of dementia (Cummings & Cotman, 1995, Lancet 326:1524-1587). While some opinion holds that amyloid plaques are a late stage by-product of the disease process, the consensus view is that amyloid plaques and/or soluble aggregates of amyloid peptides are more likely to be intimately, and perhaps causally, involved in Alzheimer's disease.
A body of published experimental evidence supports this view. For example, amyloid β-protein (“Aβ”), a primary component of amyloid plaques, is toxic to neurons in culture and transgenic mice that overproduce Aβ in their brains show significant deposition of Aβ into amyloid plaques as well as significant neuronal toxicity (Yankner, 1990, Science 250:279-282; Mattson et al., 1992, J. Neurosci. 12:379-389; Games et al., 1995, Nature 373(6514):523-527; LaFerla et al., 1995, Nature Genetics 9:21-29). Mutations in the APP gene, leading to increased Aβ production, have been linked to heritable forms of Alzheimer's disease (Goate et al., 1991, Nature 349:704-706; Chartier-Harlan et al., 1991, Nature 353:844-846; Murrel et al., 1991, Science 254:97-99; Mullan et al., 1992, Nature Genetics 1:345-347). Presenilin-1 (PS1) and presenilin-2 (PS2) related familial early-onset Alzheimer's disease (FAD) shows disproportionately increased production of Aβ1-42, the 42 amino acid isoform of Aβ, as opposed to Aβ1-40, the 40 amino acid isoform (Scheuner et al, 1996, Nature Medicine 2:864-870). The longer isoform of Aβ is more prone to aggregation than the shorter isoform (Jarrett et al, 1993, Biochemistry 32:4693-4697). Injection of the insoluble, fibrillar form of Aβ into monkey brains results in the development of pathology (neuronal destruction, tau phosphorylation, microglial proliferation) that closely mimics Alzheimer's disease in humans (Geula et al., 1998, Nature Medicine 4:827-831). See Selkoe, 1994, J. Neuropathol. Exp. Neurol. 53:438-447; Hardy and Selkoe, 2002, Science 297 (5580) 353-6; for reviews of the evidence that amyloid plaques have a central role in Alzheimer's disease.
APP is a ubiquitous membrane-spanning (type 1) glycoprotein that undergoes a variety of proteolytic processing events. (Selkoe, 1998, Trends Cell Biol. 8:447-453). APP is actually a family of polypeptides produced by alternative splicing from a single gene. Major forms of APP are known as APP695, APP751, and APP770, with the subscripts referring to the number of amino acids in each splice variant (Ponte et al., 1988, Nature 331:525-527; Tanzi et al., 1988, Nature 331:528-530; Kitaguchi et al., 1988, Nature 331:530-532).
Aβ, a 38-43 amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP), is the major component of amyloid plaques (Glenner & Wong, 1984, Biochem. Biophys. Res. Comm. 120:885-890). APP is expressed and constitutively catabolized in most cells. APP has a short half-life and is metabolized rapidly down two pathways. In one pathway, cleavage by an enzyme known as α secretase occurs while APP is still in the trans-Golgi secretory compartment (Kuentzel et al., 1993, Biochem. J. 295:367-378). This cleavage by α secretase occurs within the Aβ portion of APP, thus precluding the formation of Aβ.
In contrast to this non-amyloidogenic pathway involving α secretase described above, proteolytic processing of APP by β secretase exposes the N-terminus of Aβ, which after γ secretase cleavage at the variable C-terminus, liberates Aβ. This Aβ-producing pathway involves cleavage of the Met671-Asp672 bond (numbered according to the 770 amino acid isoform) by β secretase. The C-terminus is actually a heterogeneous collection of cleavage sites rather than a single site since γ secretase activity occurs over a short stretch of APP amino acids rather than at a single peptide bond. Peptides of 40 or 42 amino acids in length (Aβ1-40 and Aβ1-42, respectively) predominate among the C-termini generated by γ secretase, however, a recent report suggests 1-38 is a dominant species in cerebrospinal fluid. (J. Wiltfang et al, unpublished, presented at International Congress of Alzheimer Disease, July 2002, Stockholm, Sweden). Aβ1-42 is more prone to aggregation than Aβ1-40, the major component of amyloid plaque (Jarrett et al., 1993, Biochemistry 32:4693-4697; Kuo et al., 1996, J. Biol. Chem 271:4077-4081), and its production is closely associated with the development of Alzheimer's disease (Sinha & Lieberburg, 1999, Proc. Natl. Acad. Sci. USA 96:11049-11053). The bond cleaved by γ secretase appears to be situated within the transmembrane domain of APP. It is unclear as to whether the C-termini of Aβ1-40 and A 1-42 are generated by a single γ secretase protease with relaxed specificity or by two distinct proteases. For a review that discusses APP and its processing, see Selkoe, 1998, Trends Cell. Biol. 8:447-453. In the amyloidogenic pathway, APP is cleaved by β secretase to liberate sAPPβ and CTFβ, which CTFβ is then cleaved by γ secretase to liberate the harmful Aβ peptide.
While abundant evidence suggests that extracellular accumulation and deposition of Aβ is a central event in the etiology of AD, recent studies have also proposed that increased intracellular accumulation of Aβ or amyloid containing C-terminal fragments may play a role in the pathophysiology of AD. For example, over-expression of APP harboring mutations which cause familial Aβ results in the increased intracellular accumulation of CTFβ in neuronal cultures and Aβ42 in HEK 293 cells. Aβ42 is the 42 amino acid long form of Aβ that is believed to be more potent in forming amyloid plaques than the shorter forms of Aβ. Moreover, evidence suggests that intra- and extracellular Aβ are formed in distinct cellular pools in hippocampal neurons and that a common feature associated with two types of familial AD mutations in APP (“Swedish” and “London”) is an increased intracellular accumulation of Aβ42. Thus, based on these studies and earlier reports implicating extracellular Aβ accumulation in AD pathology, it appears that altered APP catabolism may be involved in disease progression.
Of key importance in this Aβ-producing pathway is the position of the γ secretase cleavage. If the γ secretase proteolytic cut is at residue 711-712, short Aβ (Aβ40) is the result; if it is a proteolytic cut after residue 713, long Aβ (Aβ42) is the result. Thus, the γ secretase process is central to the production of Aβ peptide of 40 or 42 amino acids in length (Aβ40 and Aβ42, respectively). For a review that discusses APP and its processing, see Selkoe, 1998, Trends Cell. Biol. 8:447-453; Selkoe, 1994, Ann. Rev. Cell Biol. 10:373-403. See also, Esch et al., 1994, Science 248:1122.
Reports show that soluble β-amyloid peptide is produced by healthy cells into culture media (Haass et al., 1992, Nature 359:322-325) and in human and animal CSF (Seubert et al., 1992, Nature 359:325-327).
Cleavage of APP can be detected in a number of convenient manners, including the detection of polypeptide or peptide fragments produced by proteolysis. Such fragments can be detected by any convenient means, such as by antibody binding. Another convenient method for detecting proteolytic cleavage is through the use of a chromogenic β secretase substrate whereby cleavage of the substrate releases a chromogen, e.g., a colored or fluorescent, product. More detailed analyses can be performed including mass spectroscopy.
As noted above, various naturally occurring mutations in APP have been identified that lead to familial, early-onset Alzheimer's disease. Once such mutation, known as the “Swedish” mutation, consists of a double change in the amino acid sequence of APP695 at the β secretase cleavage site: K595, M596 to N595, L596 (Mullan et al., 1992, Nature Genet. 1:345; Citron et al., 1992, Nature 360:672). cultured cells that express a cDNA encoding APP bearing the Swedish version of the β secretase cleavage site produce about 6-8 fold more Aβ than cells expressing wild-type APP (Citron et al., 1992, Nature 360:672-674).
Citron et al., 1995. Neuron 14:661-670 varied the amino acid sequence at the β secretase cleavage site of APP (positions Val594-Ala598 of APP695) and found that most substitutions in this sequence strongly decreased or eliminated cleavage by β secretase. Only the Swedish mutation was found to strongly increase cleavage.
Sisodia, 1992, Proc. Natl. Acad. Sci. USA 89(13): 6075-6079.described experiments in which various changes in the amino acid sequence of APP in the region of the βsecretase cleavage site were made and the effect of those changes on cleavage by βsecretase were measured. A change of K to V at position 612 of the 695 amino acid version of APP led to reduced cleavage by βsecretase. The K612V change has been built into a vector encoding the carboxy terminal 99 amino acids of APP and transgenic mice expressing this construct have been obtained. Such mice develop a myopathy similar to human inclusion body myositis (Jin et al., 1998, Am. J. Pathol. 153:1679-1686).
Much interest has focused on the possibility of inhibiting the development of amyloid plaques as a means of preventing or ameliorating the symptoms of Alzheimer's disease. To that end, a promising strategy is to inhibit the activity of β- and γ secretase, the two enzymes that together are responsible for producing Aβ. This strategy is attractive because, if amyloid plaque formation as a result of Aβ deposition is a cause of Alzheimer's disease, then inhibiting the activity of one or both of the two secretases would intervene in the disease process at an early stage, before late-stage events such as inflammation or apoptosis occur. Such early stage intervention is expected to be particularly beneficial (see, e.g., Citron, 2000, Molecular Medicine Today 6:392-397).
To that end, various assays have been developed that are directed to the identification of substances that may interfere with the production of Aβ or its deposition into amyloid plaques. U.S. Pat. No. 5,441,870 is directed to methods of monitoring the processing of APP by detecting the production of amino terminal fragments of APP. U.S. Pat. No. 5,605,811 is directed to methods of identifying inhibitors of the production of amino terminal fragments of APP. U.S. Pat. No. 5,593,846 is directed to methods of detecting soluble Aβ by the use of binding substances such as antibodies. Esler et al., 1997, Nature Biotechnology 15:258-263 described an assay that monitored the deposition of Aβ from solution onto a synthetic analogue of an amyloid plaque. The assay was suitable for identifying substances that could inhibit the deposition of Aβ. However, this assay is not suitable for identifying substances, such as inhibitors of β- or γ secretase, that would prevent the formation of Aβ.
Various groups have cloned and sequenced cDNA encoding a protein that is believed to be β secretase (Vassar et al., 1999, Science 286:735-741; Hussain et al., 1999, Mol. Cell. Neurosci. 14:419-427; Yan et al., 1999, Nature 402:533-537; Sinha et al., 1999, Nature 402:537-540; Lin et al., 2000, Proc. Natl. Acad. Sci. USA 97:1456-1460). Hong et al., 2000, Science 290:150-153 determined the crystal structure of the protease domain of human β secretase complexed with an eight-residue peptide-like inhibitor at 1.9 angstrom resolution. Compared to other human aspartyl proteases, the active site of human β secretase is more open and less hydrophobic, contributing to the broad substrate specificity of human β secretase (Lin et al., 2000, Proc. Natl. Acad. Sci. USA 97:1456-1460).
Ghosh et al., 2000, J. Am. Chem. Soc. 122:3522-3523 disclosed two inhibitors of β secretase, OM99-1 and OM99-2, that are modified peptides based on the β secretase cleavage site of the Swedish mutation of APP (SEVNL/DAEFR (SEQ ID NO: 1), with “/” indicating the site of cleavage). OM99-1 has the structure VNL*AAEF (SEQ ID NO: 2) (with “L*A” indicating the uncleavable hydroxyethylene transition-state isostere of the LA peptide bond) and exhibits a Ki towards recombinant β secretase produced in E. coli of approximately 68+/−3 nM. OM99-2 has the structure EVNL*AAEF (SEQ ID NO: 3) (with “L*A” indicating the uncleavable hydroxyethylene transition-state isostere of the LA peptide bond) and exhibits a Ki towards recombinant β secretase produced in E. coli of 96±3 nM. OM99-1 and OM99-2, as well as related substances, are described in International Patent Publication WO 01/00665.
A recent report indicates that β secretase inhibitors can act at micromolar levels in cell culture (Hom et al, J. Med Chem Letters, published online JM0256191, April 2003). Despite progress in identifying β secretase inhibitors, there are currently no approved pharmaceuticals for the treatment or prevention of Alzheimer's disease that are believed to exert their therapeutic effect through the inhibition of β secretase. Thus, there remains a need for additional assays that can be used to identify additional inhibitors of β secretase.
It is well known in the art that transgenic and genetically engineered animal models are useful for both basic and applied research in the field of Alzheimer's Disease research. Many human genetic mutations have been discovered through linkage and association analysis. These human alleles have been engineered into several animal models and serve as the basis of much of the in vivo research performed in the field. Among the most prevalent animal models are the PDAPP mouse (Games et al, 1995, Nature 373(6514):523-7) which encodes the V717F mutation in APP, the Tg2576 APPsw mouse encoding the Swedish mutation in APP (Hsiao et al, 1996, Science 274 (5283):99-102.) and mutant presenilin 1 transgenics (Duff et al, 1996, Nature 24:383(6603):710-3)). Each of these mice have enabled study of aspects of the processing of APP in vivo. In particular, the Tg2576 mouse model develops plaque pathology and is now a standard model for preclinical investigations of the amyloidosis process. The T2576 mouse model is often used in conjunction with behavioral and cognitive deficit studies. In addition to the Tg2576 mouse model, several YAC models have been created to more accurately reflect the appropriate regulatory elements (Lamb et al, 1993, Nature Genetics 5(1):22-30). In addition to these gain-of-function animal models there are several loss-of-function Knock-out (KO) animal models including PS1 KO (Feng et al., 2001, Neuron 32(5):911-26), BACE1 KO (Cai et al., 2001, Nature Neuroscience (3):2334), ADAM10 KO and APP KO (Zheng et al, 1995, Cell 81(4)525-31). In addition to APP and Presenilin mutations, several groups have also created Tau transgenic animals. The P301L Tau mutant mice of Gotz et al (see Gotz et al., 2001, Science 293 (5534): 1491-5) and Lewis et al., 2000, Nature Genetics (4):402-5) are examples. Additionally, others have created α-synuclein transgenic animal models, including the A53T mutant mice (Giasson, 2002, Neuron 34(4):521-33).
Although each animal model is useful alone, they are often more useful when cross-bred in combination with other genetically engineered and transgenic models. Several prevalent cross-breeds include the Tg2576 ×PSI mouse (Duff et al. 1988, Nature Medicine 4 (1): 97-100) and the TgCRND8model encoding the combination of APPsw and V717F (Chishti et al, 2001, J. Biol. Chem. 276(24):21562-70). Moreover, there are bi-genic models which include APPsw ×P301L Tau (Lewis et al, 2001, Science 293 (5534):1487-91) and APPsw ×A53T (Masliah, 2001, Proc. Natl Acad Sci USA 98(21):12245-12250). Finally, the recent emergence of triple transgenics animals, including the APPsw ×P301L ×PS1 model (F. LaFerla et al unpublished, presented at 6th International AD/PD conference, May 8-12, 2003, Seville, Spain) and Swedish/Dutch/Iowa triple transgenic (W. B. van Nostrand, Stony Brook, unpublished, presented at 6th International AD/PD conference, May 8-12, 2003, Seville, Spain) provides the ability to work with models systems that approximate aspects of particular disease states. The bi-genic and tri-genic animals are exemplary of the possible combinations that one can create by crossing the animals of the present invention to create models relevant to finding a medical treatment for Alzheimer's Disease.
Improved animal models for human Alzheimer Disease advance both mechanistic understanding and preclinical therapeutic validations. A number of useful animal models for Alzheimer Disease have been produced but the majority do not accurately reproduce the spatial and temporal expression of amyloid precursor protein over the lifetime of the animal. Such model systems often produce supra-physiological levels of the full length amyloid substrate protein, upon which the various proteolytic enzymes operate. The overexpression of APP and the reliance of many models on heterologous promotors may possibly confound potential feedback loops that operate on the modulation of APP gene expression. To more closely model the endogenous spatial and temporal expression patterns and natural history of amyloid pathophysiology, we have used gene-targeting in mouse embryonic stem cells to modify the mouse APP protein at the endogenous APP genetic locus. Thus the present invention provides an animal model of Alzheimer Disease which exhibits a wild-type pattern of APP expression while providing important phenotypes including a significant enhancement of beta-secretase site cleavage, an increased ratio of beta-secretase activity to alpha secretase activity and elevated amyloid production.